1. Field of the Invention
This invention relates to the area of medical genetics. Specifically, the invention provides a genetic test to identify carriers of specific alleles of the Alpha-2-Macroglobulin gene, the occurrence of which are genetically linked, with high statistical significance, to the condition known as Alzheimer""s disease.
2. Related Art
Alpha-2-Macroglobulin (xcex12M) is a major serum pan-protease inhibitor which inhibits all four classes of proteases by a unique steric trapping mechanism (Borth, W., FASEB 6:3345-3353 (1992)). It is the major representative of a family of plasma proteins, most of which possess a unique internal, cyclic thiol ester bond, referred to as the xcex1-macroglobulins (Borth, W., FASEB 6:3345-3353 (1992)). Although it functions against a wide variety of human and bacterial endopeptidases (Starkey, P. M. and Barret, A. J., Proteinases in mammalian cells and tissues, Barret, A. J. (ed.), North-Holland, N.Y. (pub), pp. 663-696, (1977)), a possible role in pathogenesis is not yet known. In vitro studies have provided a great deal of information regarding protein structure and inhibitory functions (Scottrup-Jensen, L., et al., J Biol. Chem. 259:8318-8327 (1984); Feldman, S. R., et al., Proc. Natl. Acad. Sci. USA 82:5700-5704, 1985; Kan, C. C., et al., Proc. Natl. Acad Sci USA 82:2282-2286 (1985); Bretaudiere, J. P., et al., Proc. Natl. Acad. Sci. USA 85:1437-1441 (1988); Van leuven, F., et al., J Biol. Chem. 261:11369-11373 (1986)), and at the molecular level, it is known to be part of a related gene cluster on human chromosome 12p (Devriendt, K., et al., Gene 81:325-334, 1989) that also includes the xcex12M pseudogene and the pregnancy-zone gene. Recent evidence suggests that xcex12M may be associated with certain morphological characteristics of Alzheimer""s Disease (AD).
AD is a neurodegenerative disorder characterized by a global decline in mental function, memory and acquired intellectual skills. It is the most common form of dementia occurring in mid-to late-life and is a major cause of disability and death in the elderly (Price, L. and Sisodia, S., Annu. Rev. Neurosci. 21:479-505 (1998)). The appearance of AD in the population according to age is variable, often being categorized into early and late onset forms. In the general population, 40% of the brains of normal individuals show some evidence of Axcex2 deposits, indicating a subclinical prevalence, and in the population 60 years and older, AD is diagnosed in 10% of the population.
AD is a genetically heterogenous disorder. Early-onset familial AD (EO-FAD) is inherited as an autosomal dominant disorder involving defects in at least three genes, presenilin I (PSENI) on chromosome 14 (Sherrington, R., et al., Nature 375:754-760 (1995)), presenilin 2 (PSEN2) on chromosome 1 (Levy-Lehad, E., et al., Science 269:973-977 (1995); Rogaev, E. I., et al., Nature 376:775-778 (1995)), and xcex2-amyloid precursor protein (APP) on chromosome 21 (Tanzi, R. E., et al., Science 235:880-884 (1987); Goate, A. M., et al., Nature 349:704-706 (1991)). These genes account for roughly 30-40% of EO-FAD (Hardy J., Trends Neurosci. 20:154-159 (1997); Cruts, M., et al., Hum. Mol. Genet. 7.43-51 (1998); Blacker, D. and Tanzi, R. E., Arch Neurol 55:294-296 (1998)).
Late-onset AD (LOAD) has been associated with the APOE-xcex54 allele of apolipoprotein E (APOE) located on chromosome 19 (Strittmatter, W. J., et al., Proc. Natl. Acad Sci. (USA) 90:1977-1981 (1993); Saunders, A. M., et al., Neurology 43:1467-1472 (1993)). Inheritance of APOE-xcex54 lowers the age of onset of AD in a dose-dependent manner and is associated with cases of AD occurring from 40 to 90 years. However it has the greatest impact as a risk factor for onset in the 60""s (Blacker, D., et al, Neurology 48:139-147 (1997); Farrer, L. A., et al., JAMA 278:1349-1356 (1997)). More recently, the gene encoding the major neuronal receptor for apoE, the low density lipoprotein receptor-related protein (LRP), located on the long arm of chromosome 12, was shown to be associated with LOAD (Kang, D. E., et al., Neurology 49:56-61 (1997)).
Neuropathological hallmarks of AD include abundant neurofibrillary tangles (NFIT), and xcex2-amyloid deposition in cerebral blood vessels and in senile plaques (SP). Deposited amyloid is composed principally of the 40-42 residue xcex2-amyloid protein (Axcex2) (Glenner et al., Biochem, Biophys Res. Commun. 120:885-890 (1984)), which is a proteolytic fragment of the xcex2-amyloid precursor protein (APP) Wang et al., Nature 325:733-736 (1987)). Pathogenesis is thought to develop in AD patients when the Axcex2 protein is organized into neurotoxic fibrils. In vitro studies demonstrate that Axcex2 is generated as a soluble peptide during cellular metabolism (Haas, C., et al., Nature 359:322-325, 1992; Shoji, M., et al., Science 258:126-129 (1992)). Several additional plaque-associated proteins are known to promote the in vitro formation of Axcex2 fibrils, suggesting the possibility that these proteins regulate Axcex2 aggregation in vivo (Ma, J., et al., Nature 372:92-94 (1994); Eriksson, S., et al., Proc Natl. Acad. Sci. USA 92:2313-2317 (1995)). Because Axcex2 deposition likely plays a role in the development of AD, a great deal of research is focused on the synthesis, degradation and clearance of this protein from the affected tissues.
Furthermore, given the potential importance of the APP and Axcex2 proteins in AD pathogenesis, research is also focused on providing a genetic link between mutations in the APP gene and the occurrence of Alzheimer""s disease. Mutations in APP close to or in the Axcex2 domain are known (Goate et al., Nature 349:704-706 (1991); Levy et al., Science 248:1124-1126 (1990); Murrell et al., Science 254:97-99(1991); Hendricks et al., Nature Genet. 1:218-221 (1992); Chartier-Harlin, M. et al., Nature 353:844 (1991); Mullan, M., et al., Nature Genet. 1:345 (1992)). In some instances, linkage of the mutation with the occurrence of familial Alzheimer""s disease (FAD) was possible.
Also implicated in the development of AD is the possibility that there exists an imbalance between proteases and protease inhibitors which affects normal amyloid metabolism. Antibodies to ubiquitin strongly react with the characteristic amyloid neurofibrillar tangles (Tabaton, M., et al., Proc. Natl. Acad Sci. 88:2098-2102 (1991)). The Axcex2 peptide is generated proteolytically from APP, and proteases cathepsin B and D and protease inhibitors such as protease nexin 1 and xcex11 -antichymotrypsin were found in amyloid plaques (Cataldo, A. M. and Nixon, R. A., Proc. Natl. Acad Sci. 87:3861-3865, 1991; Rosenblatt, D. E., et al., Ann. Neurol 26:628-634 (1989); Abraham, C. R., et al., Cell 52:487-501 (1988)).
Alpha-2-macroglobulin is another protease inhibitor implicated in neural tissue metabolism. Several lines of evidence link the xcex12M protein to morphological properties associated with AD patient cerebra. One study in support of a role in neural metabolism identified xcex12M as an astroglia-derived neurite-promoting factor for cultured neurons from the rat central nervous system (Mori, T., et al., Brain Res. 527:55-61 (1990)). In addition, xcex12M has been shown to attenuate the fibrillogenesis and neurotoxicity of Axcex2 (Hughes, S. R., et al., Proc. Natl. Acad. Sci. (USA) 95:3275-3280 (1998); Du, Y., et al., J Neurochem. 70:1182-1188 (1998); Zhang, Z., et al., Amyloid: Int. J Exp. Clin. Invest. 3:156-161 (1996)). Also suggestive of a connection between xcex12M and AD is that xcex12M tightly binds the Axcex2 peptide (Du, Y., et al, J Neurochem. 69:299-305 (1997); Hughes, S. R., et al., Proc. Natl. Acad Sci. (USA) 95:3275-3280 (1998)), and has been shown to mediate the clearance of Axcex2 via endocytosis through LRP (Narita, M., et al., J Neurochem. 69: 1904-1911 (1997)). Furthermore, two independent studies found that an xcex12M/protease complex is capable of degrading the Axcex2 peptide (Zhang, Z., et al., Amyloid: Int. J. Exp. Clin. Invest. 3:156-161 (1996); Qiu, W. Q., et al., J. Biol. Chem. 271(14):8443-8451 (1996)). In addition, xcex12M has been localized to senile plaques (SP) in AD (Bauer, J., et al., FEBS 285:111-114 (1991); Van Gool, D., et al., Neurobiology of Aging 14:233-237 (1993); Rebeck, G. W., et al., Ann. Neurol 37:211-217 (1995)). Van Gool et al., suggest that expression of xcex12M could be associated with neurofibrillary changes in senile plaques in AD. (Van Gool, D., et al., Neurobiology of Aging 14:233-237 (1993)). An immunocytochemistry study of cerebra of AD patients using two different monoclonal antibodies specific for xcex12M found localization in neuritic-type plaques but not preamyloid or burned-out type plaques (Van Gool, D., et al., Neurobiology of Aging 14:233-237 (1993)). Because immunoreactivity was associated with microglia in the outer border of the neuritic plaque, Van Gool et al. suggest that xcex12M could be a marker for an inflammatory process in the plaques (Van Gool, D., et al., Neurobiology of Aging 14:233-237 (1993)).
The human xcex12M gene has been cloned and several mutants have been identified. (Poller, W., et al., Human Genetics 88:313-319 (1992); Matthijs, G., and Marynen, P., Nucl. Acids Res. 19(18):5102 (1991)). One mutation represented a bi-allelic deletion polymorphism in which 5 bases were deleted at positions xe2x88x927 to xe2x88x923 of the 5xe2x80x2 splice acceptor site of exon 18 (Matthijs, G., and Marynen, P., Nucl Acids Res. 19(18):5102 (1991)) (referred to as the A2M-2 allele). The A2M-2 allele, and the wild type A2M allele (referred to as the A2M-1 allele) were found at allele frequencies of 0.18 and 0.82 respectively (Matthijs, G., and Marynen, P., Nucl. Acids Res. 19(18):5102 (1991)). The biological consequences of this deletion mutation have not yet been reported, but it is known that exon 18 encodes xe2x80x9cexon IIxe2x80x9d of the bait domain of xcex12M, which is used to trap proteases.
Another mutation represented a simple polymorphism 25 amino acids downstream from the thiolester site of the protein that interchanges Val1000(GTC) and Ile1000 (ATC) (Poller, W., et al., Human Genetics 88:313-319 (1992) (numbering is based on the cDNA sequence which includes a 24 amino acid signal peptide; this amino acid corresponds to Val/Ile976 in the mature protein). The Val, or G allele, and the Ile, or A allele, were found at allele frequencies of 0.30 and 0.70, respectively (Poller, W., et al., Human Genetics 88:313-319 (1992)). No difference in xcex12M serum levels was associated with the two alleles. This polymorphism is interesting because the mutation occurs near the thiolester active site of the molecule.
Family, twin, and population data all suggest that genes involved in AD remain to be identified (Lautenschlager, N. T., et al., Neurology 46(3):641-50 (1996); Bergem, A., et al., Arch. Gen. Psychiatry 54:264-270 (1997); Payami, H., et al., Am J Hum Genet 60:948-956 (1997)) and several candidates have been reported (for a review, see Hardy J., Trends Neurosci. 20:154-159 (1997); Cruts, M., et al., Hum. Mol. Genet. 7:43-51 (1998); and Blacker, D., and Tanzi, R. E., Arch. Neurol. 55:294-296 (1998)). The identification of specific genes or alleles thereof linked to the development of AD will provide a greater understanding of the mechanisms behind the disease process, a screening method for detection of at-risk individuals, and may lead to effective treatment of this disease. A great deal towards this end has been accomplished already in the study of the APP gene. This invention, linking A2M, and particularly inheritance of the A2M-2 and A2M-G alleles, to the occurrence of Alzheimer""s disease, provides another important resource.
This invention relates to the discovery that particular alleles of A2M are linked to the occurrence of Alzheimer""s disease. More specifically, it was found that individuals carrying at least one copy of the A2M-2 allele or who are homozygous for the A2M-G allele are disproportionately represented in a population of AD patients as compared to those unaffected by AD.
Based on this discovery, a diagnostic method was developed that characterizes the A2M genotype of individuals in the population to assess risk for developing AD. In one embodiment of the invention, A2M genotype is determined by isolating nucleic acid from an individual, and analyzing it for the particular A2M alleles of interest. Various embodiments of the invention enable analysis from isolated nucleic acid that is DNA or RNA. In a preferred embodiment, restriction fragment length polymorphism (RFLP) analysis is used to determine A2M genotype.
In a more preferred embodiment of the invention, the nucleic acid is utilized as a template for the amplification of an A2M gene fragment prior to the genotyping analysis. A preferred method is to amplify a fragment that contains the site of the pentanucleotide deletion mutation found in the A2M-2 allele. Analysis of the product of amplification is preferably accomplished by determining the polynucleotide sequence of the amplified fragment. Other methods of analyzing the amplified fragment include size determination, single-strand conformation polymorphism (SSCP) analysis, and RFLP analysis. Another preferred method is to amplify a fragment that encodes amino acid 1000, the residue affected by the mutation found in the A2M-G allele. Analysis of the product of amplification is preferably accomplished by RFLP analysis. Other methods of analyzing the amplified fragment include sequencing or sizing the fragment, and SSCP analysis.
Another embodiment of the invention relates to the use of protein isotyping to assess A2M genotype . In one embodiment, an antibody specific for an xcex12M variant lacking exon 18 (xcex12M-2) is used to assess A2M genotype through protein isotyping. In a preferred method of protein isotyping, a second antibody, which is specific for an a xcex12M protein having exon 18 (the wild type xcex12M referred to as xcex12M-1), is used as a control. Yet another embodiment of the invention relates to the use of an antibody specific for the Val1000 variant to assess the A2M-G genotype through protein isotyping. In a preferred method of protein isotyping, a second antibody, which is specific for the Ile1000 protein, is used as a control. For the protein isotyping methods of the invention, preferably, protein is isolated from an individual and screened with the specific antibodies. More specifically, a more preferred embodiment would utilize Western blot or ELISA technology. In yet another embodiment of the invention an xcex12M electrophoretic mobility assay is used to detect the xcex12M-2 variant to assess A2M genotype through protein isotyping.
The invention also relates to diagnostic kits for A2M genotyping. Alternative embodiments of the genotyping kit enable screening of nucleic acid or protein, providing a fast and efficient means for Alzheimer""s disease risk assessment.